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On‐plate digestion of proteins using novel trypsin‐immobilized magnetic nanospheres for MALDI‐TOF‐MS analysis
Author(s) -
Li Yan,
Yan Bo,
Deng Chunhui,
Tang Jia,
Liu Junyan,
Zhang Xiangmin
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700464
Subject(s) - trypsin , chemistry , digestion (alchemy) , chromatography , peptide , magnetic nanoparticles , sample preparation , amine gas treating , myoglobin , biochemistry , enzyme , materials science , nanotechnology , organic chemistry , nanoparticle
In this study, a novel method of on‐plate digestion using trypsin‐immobilized magnetic nanospheres was developed followed by MALDI‐TOF‐MS for rapid and effective analysis and identification of proteins. We utilized a facile one‐pot method for the direct preparation of amine‐functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin‐linked magnetic nanospheres were then applied for on‐plate digestion of sample proteins (myoglobin and Cytochrome c ). Moreover, after digestion, the trypsin‐linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in‐solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on‐plate digestion protocol for MALDI‐MS may find further application in automated analysis of large sets of proteins.